Hadwan Mahmoud Hussein, Hussein Marwah Jaber, Mohammed Rawa M, Hadwan Asad M, Saad Al-Kawaz Hawraa, Al-Obaidy Saba S M, Al Talebi Zainab Abbas
Department of Chemistry, College of Science, University of Babylon, Hilla 51002, Iraq.
Department of Medical Physics, University of Al-Mustaqbal, Hilla 51001, Iraq.
Biol Methods Protoc. 2024 Mar 5;9(1):bpae015. doi: 10.1093/biomethods/bpae015. eCollection 2024.
Catalase (CAT) is an important enzyme that protects biomolecules against oxidative damage by breaking down hydrogen peroxide (HO) into water and oxygen. CAT is present in all aerobic microbes, animals, and plants. It is, however, absent from normal human urine but can be detected in pathological urine. CAT testing can thus help to detect such urine. This study presents a novel spectrophotometric method for determining CAT activity characterized by its simplicity, sensitivity, specificity, and rapidity. The method involves incubating enzyme-containing samples with a carefully chosen concentration of HO for a specified incubation period. Subsequently, a solution containing ferrous ammonium sulfate (FAS) and sulfosalicylic acid (SSA) is added to terminate the enzyme activity. A distinctive maroon-colored ferrisulfosalicylate complex is formed. The formation of this complex is a direct result of the reaction between FAS and any residual peroxide present. This leads to the generation of ferric ions when coordinated with SSA. The complex has a maximum absorbance of 490 nm. This advanced method eliminates the need for concentrated acids to stop CAT activity, making it safer and easier to handle. A comparative analysis against the standard ferrithiocyanate method showed a correlation coefficient of 0.99, demonstrating the new method's comparable effectiveness and reliability. In conclusion, a simple and reliable protocol for assessing CAT activity, which utilizes a cuvette or microplate, has been demonstrated in this study. This interference-free protocol can easily be used in research and clinical analysis with considerable accuracy and precision.
过氧化氢酶（CAT）是一种重要的酶，它通过将过氧化氢（H₂O₂）分解为水和氧气来保护生物分子免受氧化损伤。CAT存在于所有需氧微生物、动物和植物中。然而，正常人类尿液中不存在CAT，但在病理性尿液中可以检测到。因此，CAT检测有助于检测此类尿液。本研究提出了一种测定CAT活性的新型分光光度法，其特点是简单、灵敏、特异且快速。该方法包括将含酶样品与精心选择浓度的H₂O₂在指定的孵育期内孵育。随后，加入含有硫酸亚铁铵（FAS）和磺基水杨酸（SSA）的溶液以终止酶活性。形成一种独特的栗色铁磺基水杨酸络合物。这种络合物的形成是FAS与任何残留过氧化物之间反应的直接结果。这在与SSA配位时会导致铁离子的产生。该络合物在490nm处有最大吸光度。这种先进的方法无需使用浓酸来停止CAT活性，使其更安全且更易于操作。与标准硫氰酸铁法的比较分析显示相关系数为0.99，表明新方法具有相当的有效性和可靠性。总之，本研究展示了一种使用比色皿或微孔板评估CAT活性的简单可靠方案。这种无干扰方案可以很容易地用于研究和临床分析，具有相当高的准确性和精密度。
